Diverse attempts to improve the efficiency of the evaluation of fluorescent samples are known in prior-art confocal fluorescence scanning microscopy, e.g., in DE 197 02 753 A1.
Solutions in this regard diverge in their aims which are directed to:                a) reducing measuring times in spite of scanning sequential image formation; and        b) blur-free imaging through an optical section, i.e., separating out-of-focus fluorescent light from the usable focal signal through a confocal aperture.        
Further, the known advantages of laser scanning microscopy (confocal microscopy) such as optical sectioning (suppression of out-of-focus signals) and high flexibility are accompanied by disadvantages and limitations:                1) expenditure of time on sequential data acquisition;        2) damage to sample (e.g., bleaching) through high intensity in the focus; and        3) damage to sample (e.g., bleaching) through unused out-of-focus intensity.        
The simplest solution for both of the above-mentioned aims a) and b), which would consist in increasing the intensity of the excitation light is not feasible as a singular approach because it is accompanied by an unwanted bleaching of the sample. Therefore, aim a) is chosen, e.g., through parallelization of measurements (multi-spot LSM, spinning disc), i.e., the simultaneous scanning of the sample with a plurality of foci which are then imaged through an own confocal pinhole or shared confocal pinhole onto a detector element or a detector array (camera, spinning disc). Accordingly, either the recording speed is increased or a larger surface area is scanned over the same time. Thus, at a given image capture rate, the second disadvantage can also be reduced in that the selected focal intensity can be reduced by the factor of parallelization. However, nothing changes with respect to the “waste” of out-of-focus photons (third disadvantage). This last aspect—apart from the unwanted stress on the sample—represents a fundamental limitation of the confocal method in that many planes of the sample are illuminated but are not evaluated although the integrated excitation output is the same in every plane.
Possibilities for reducing stressing of samples through parallelized image capture are described, for example, in U.S. Pat. No. 5,239,178 or U.S. Pat. No. 6,028,306 with N separate measuring volumes (referred to as measuring points for the sake of simplicity) which are illuminated and measured simultaneously in the focal plane. The sample can be measured with less intensity per illumination beam at the same time at N points. The illumination intensity is reduced by the factor 1/N and the pixel dwell time is lengthened by the factor N so that the frame rate is identical to, and the SNR is comparable to, the raster scanned recording by means of an individual measuring volume. While the energy dose entering the sample is the same, it is spatially distributed such that the peak intensity which is harmful to the sample can be reduced per illumination point.
An image capture with the same advantages which is parallel in many respects can also be accomplished by means of a rotating Nipkow disc or with linear scanning.
An alternative kind of parallelized image capture consists in simultaneous imaging of measuring points from different image planes, also known as axial multifocal imaging.
For example, it is known from DE 103 56 416 A1 to achieve a simultaneous imaging of a plurality of separate measuring volumes located along the optical axis of the microscope objective based on monochromatic confocal microscopy by means of an optically decentered diffractive optical element (DOE, e.g., phase grating) and collector optics in that the different curvature of wavefronts originating from sample planes at different distances from one another is used to distribute them through the DOE in different diffraction orders and to image them in an individual plane, preferably the confocal aperture plane. All of the wavelengths except that of the illumination light are then discriminated with a confocal aperture, so this multifocal imaging variant is not suitable for florescence measurements (because of Stokes shift and the spectral bandwidth of the florescence emission).
Systems for multifocal imaging in wide-field microscopy work on the same principle as described, for example, by: Blanchard et al. (1999), “Simultaneous multiplane imaging with a distorted diffraction grating”, Appl. Opt. 38 (32): 6692-6699; Dalgarno et al. (2010), “Multiplane imaging and three-dimensional nanoscale particle tracking in biological microscopy”, Optics Express 18 (2): 877-884; and Abrahamsson et al. (2013), “Fast multicolor 3D imaging using aberration-corrected multifocus microscopy”, Nature Methods 10 (1): 60-63.
Prior-art fluorescence microscopy also includes methods of fluorescence correlation analysis of which SOFI (Superresolution Optical Fluctuation Imaging) is mentioned in particular. This method is described in the following publications:                WO 2010/141608 A1;        Dertinger, T.; Colyer, R.; Iyer G.; Weiss, S.; Enderlein, J. (2009). “Fast, background-free, 3D superresolution optical fluctuation imaging (SOFI)”. In PNAS 106 (52): 22287-92;        Dertinger, T.; Colyer, R.; Vogel, R.; Enderlein, J.; Weiss, S. (2010). “Achieving increased resolution and more pixels with Superresolution Optical Fluctuation Imaging (SOFI)”, Optics Express 18 (18): 18875-84;        Geissbuehler, S.; Dellagiacoma, C.; Lasser, T. (2011). “Comparison between SOFI and STORM”, Biomed. Opt. Express 2 (3): 408-420.        
In wide-field fluorescence detection (direct imaging of a plane of the sample by means of a camera), fluctuations of fluorescence emitters are evaluated by means of SOFI with a defined temporal correlation in order to obtain a fluorescence imaging with increased resolution over the diffraction limit. The degree to which resolution is increased depends on the order of the correlation function that can be evaluated. The latter in turn is heavily dependent upon the fluctuating system and signal quality. Necessary prerequisites for the application of SOFI are:                the fluorescing system (molecule) must have at least two distinguishable fluorescence states (e.g., ON/OFF);        different emitters (molecules) must change, or “blink”, independently from one another and stochastically between these states; and        the switching between states must be temporally detectable by an image sensor (area detector).        
The first two prerequisites are met in principle for a large number of (basically all) fluorescing molecules (organic dyes and proteins); the OFF state can be, for example, a triplet state (according to FIG. 1 in Widengren, J. (2010). “Fluorescence-based transient state monitoring for biomolecular spectroscopy and imaging”, J. R. Soc. Interface 7 (49): 1135-1144) or other non-radiatively decaying state. It is also conceivable that radiative relaxation pathways can be discriminated via the wavelength. However, to date, SOFI has only been able to be demonstrated for specific fluorescence systems because the time scales within which the triplet OFF state occurs are much too short even for the fastest available continuous-operation area detectors (CMOS, CCD). The corresponding times can be read, for example, off a typical measurement curve of a Jablonski diagram (see FIG. 1 in J. Widengren, op. cit.). A three-state model of a fluorophore and an FCS (Fluorescence Correlation Spectroscopy) are shown in the latter as (a) and (c), respectively, where the lifetime of the triplet state tT is in the microsecond range.
Examples of specific systems which are detectable, however, are quantum dots (Dertinger et al. [2009] PNAS) which exhibit blinking on almost all time scales and dSTORM systems (Geissbühler et al. [2011] Opt Express) in which the blinking behavior of the emitters is rendered in time intervals that can be detected by cameras through the adaptation of the chemical environment and of the excitation conditions that is known from the dSTORM method. Therefore, SOFI and dSTORM methods are not suitable for the broad application of common fluorescent dyes.
Hereinafter, the word “dyes” will be used for both endogenous (autofluorescence) and exogenous fluorescent dyes as well as for fluorescing proteins which exhibit the temporal behavior required for the method.
Based on a fluorescence scanning microscope in the form of a multi-confocal laser scanning microscope, e.g., according to the not-prior-published DE 10 2014 002 328, with first diffractive optics arranged in the observation beam path between a beam combiner and the image plane for splitting light beams into beam bundles of different diffraction orders which have different spherical phases relative to one another, with second diffractive optics for compensation of chromatic aberrations generated through the first diffractive optics, and with collector optics for focusing the split beam bundles in the image plane so that a series of different disjoint measuring volumes arranged along the optical axis of the microscope objective is imaged on the object side in the image plane simultaneously (along the different diffraction orders of the diffractive optics), the problem remains in multi-confocal detection with the laser scanning microscope described above that confocal detection would have to be carried out again on every sensor in order to acquire an image with optical depth resolution (known as sectioning). In this respect, it does not matter whether the light passes through a physical pinhole or is filtered confocally through utilization of the pixel-per-depth plane separation on the sensor. For this reason, however, it is to be expected as disadvantageous that the detection is in no way more light-efficient than in the sequential mode of sectioning with confocal fluorescence microscope.
The “light losses” can be explained by the fact that when light is split with chirped gratings, light also proceeds in diffraction orders which image “out-of-focus”. An effect of this kind equates to the effect when using neutral splitters as described, for example, by Dalgarno et al. (2010) Optics Express 18 (2): 881, FIG. 2. In this case, a plane is sharply imaged with only a portion of the light, which is given by the splitting ratio of the beamsplitter, and the input intensity in the splitting ratio is consequently diminished. The splitting of the light with chirped gratings acts in exactly the same way as if observing with N neutral splitters in N different planes. While this makes the multi-(con)focal arrangement faster (parallelization in Z direction), the light arriving out-of-focus at the respective pinhole from other planes would be lost with confocal detection on a sensor element. But the goal of a parallelized arrangement should be to evaluate all of the light of the emitters from the respective conjugate sensor planes (i.e., from those planes of the sample that are sharply imaged by the respective sensor element).
If there were only one luminescent particle present within the excitation and detection PSFs in a so-called elongated focus range, the signal could be associated with the correct location in the sample by simultaneous non-confocal measurement of all of the planes with a pixelated sensor.
In this way, a “quasi-confocal image” would be achieved with increased efficiency, this image being generated in a manner basically corresponding to the procedure in a three-dimensional deconvolution in which the measured 3D light distribution is distributed among different sensors. However, in real measurements, an individual luminescent particle is virtually never assumed so that, in real samples, the signals from planes which are offset in the direction of the optical axis are superposed on one another on the sensor segments and, in case of a sample structure which is unknown a priori, can no longer be unequivocally associated with the planes because there is substantial crosstalk of signals from “blurrily imaged” planes on the detectors for diffraction orders separated through the DOE system owing to the absence of a confocal discrimination of signals from different planes. Moreover, the PSF is normally symmetrical so that, without further information, the portions which are defocused “up” and “down” could not be differentiated.